1. Field of the Invention
The invention relates to a gene promoter characterized in that it can be over-expressed in various plant tissues, as well as to applications of said promoter.
2. Description of the Prior Art
Banana (Musa spp.) belongs to Musaceae, Monocotyledon, with its fruit being fragrant and delicious, as well as having high nutritional value. The desired banana protein can be used to produce in large quantities vaccines useful for treating diseases in humans or animals, by using the banana as gene transfer material. The banana can be used to directly uptake the vaccine orally, which is not only more convenient and safer than a vaccine injection, but also prevents proteins from losing their function through denaturizing in cooking, since banana fruit can be eaten raw.
In order to enable a transgenic plant to produce protein in large amounts, in the construction of a gene transfer construct selection of a proper promoter for activating the target gene that is highly associated with the production of proteins is important. At present, most conventional technologies for plant gene transfer has use cauliflower mosaic virus 35S (CaMV 35S) promoter to induce the over-expression of target genes in dicotyledon or monocotyledon plants. However, the expression of CaMV 35S promoter in a monocotyledon plant is relatively low. In order to improve the activation ability of a promoter, cloning of a promoter with high expression strength originally present in the monocotyledon plant, such as the Act1 promoter for paddy rice actin gene, Ubi1 promoter for corn polyubiquitin gene, and the like, might be carried out. Among these, the expression strength of Ubi1 promoter in a monocotyledon plant system is higher and more stable than that of CaMV 35S promoter, thereby the gene expression can be increased significantly (Christensen et al., 1992; McElroy et al., 1991).
Ubiquitin is a small molecular protein extensively present in a eukaryote. Its gene belongs to a polygene family. Based on its gene structure, it can be classified into three major groups: polyubiquitin gene, ubiquitin extension gene and ubiquitin-like gene. Polyubiquitin gene consists of arranging identical mono ubiquitin gene head-to-tail in a manner of tandem repeat in same direction. There are many types of polyubiquitin gene in plants, each with different modulation mechanisms. Further, there are different expression patterns such as constitutive expression, inducible expression, tissue specific expression and the like. Polyubiquitin gene with constitutive expression can be over-expressed in all tissues and organs, such as parsley ubi 4-2 (Kawalleck et al., 1993) and UBQ10 of Arabidopsis thialana (Sun et al., 1997) and the like.
Another feature of polyubiquitin gene is that there is a first intron connected tightly with ATG at its 5′-untranslation region, where the sequence at the joint of this intron and exon is highly conserved, which is consistent with AG/gt principle at 5′-terminal cleavage and the ag/GT principle at 3′-terminal cleavage, such as genes of rice RUBQ1 and 2, sunflower UbB1 and 2 as well as Arabidopsis thialana UBQ3, 10 and 11. The presence or absence of the first intron might influence the activity of a polyubiquitin gene promoter, and its length varies with species (Norris et al., 1993; Plesse et al., 2001).
Many researches had shown that ubiquitin gene promoters isolated from a variety of plants exhibited expression activity in gene transfer system stronger than that of CaMV 35S promoter. Accordingly, studies on the activity and tissue-specific expression of ubiquitin gene promoter in order to assess its applications will become a topic worthy of future investigation.
In view of the importance of developing promoters with high activating ability for the biotechnical industry, the inventor had thought to improve and innovate, and finally, after studying intensively for many years, successfully developed the promoter with high expression strength and capable of being over-expressed in various tissues of a plant, as well as its application according to the invention.